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Glutamate Synthase: Structural, Mechanistic and Regulatory Properties, and Role in the Amino Acid Metabolism

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Glutamate Synthase: Structural, Mechanistic and Regulatory Properties, and Role in the Amino Acid Metabolism Photosynthesis Research (2005) 83: 191–217 Ó Springer 2005 Review Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism Akira Suzuki1,* & David B. Knaff2 1Unite´ de Nutrition Azote´e des Plantes, Institut National de la Recherche Agronomique, Route de Saint-Cyr, 78026 Versailles cedex, France; 2Department of Chemistry and Biochemistry, Texas Tech University, P.O. Box 41061, Lubbock, TX 79409-1061, USA; *Author for correspondence (e-mail: [email protected]; fax: +33-1-30833096) Received 28 June 2004; accepted in revised form 20 September 2004 Key words: ammonium assimilation, glutamate synthase, glutamine synthetase, higher plants, nitrogen metabolism Abstract Ammonium ion assimilation constitutes a central metabolic pathway in many organisms, and glutamate synthase, in concert with glutamine synthetase (GS, EC 6.3.1.2), plays the primary role of ammonium ion incorporation into glutamine and glutamate. Glutamate synthase occurs in three forms that can be dis- tinguished based on whether they use NADPH (NADPH-GOGAT, EC 1.4.1.13), NADH (NADH-GO- GAT, EC 1.4.1.14) or reduced ferredoxin (Fd-GOGAT, EC 1.4.7.1) as the electron donor for the (two- electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate. The distribution of these three forms of glutamate synthase in different tissues is quite specific to the organism in question. Gene structures have been determined for Fd-, NADH- and NADPH-dependent glutamate synthases from different organisms, as shown by searches in nucleic acid sequence data banks. Fd-glutamate synthase contains two electron-carrying prosthetic groups, the redox properties of which are discussed. A description of the ferredoxin binding by Fd-glutamate synthase is also presented. In plants, including nitrogen-fixing legumes, Fd-glutamate synthase and NADH-glutamate synthase supply glutamate during the nitrogen assimilation and translocation. The biological functions of Fd-glutamate synthase and NADH-glutamate synthase, which show a highly tissue-specific distribution pattern, are tightly related to the regulation by the light and metabolite sensing systems. Analysis of mutants and transgenic studies have provided insights into the primary individual functions of Fd-glutamate synthase and NADH-glutamate synthase. These studies also provided evidence that glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) does not represent a signif- icant alternate route for glutamate formation in plants. Taken together, biochemical analysis and genetic and molecular data imply that Fd-glutamate synthase incorporates photorespiratory and non-photore- spiratory ammonium and provides nitrogen for transport to maintain nitrogen status in plants. Fd-glu- tamate synthase also plays a role that is redundant, in several important aspects, to that played by NADH- glutamate synthase in ammonium assimilation and nitrogen transport. Abbreviations: ASN – asparagine synthetase gene; CD – circular dichroism; EPR – electron paramagnetic resonance; DG – free energy change; DS – entropy change; FAD – flavin adenine dinucleotide; Fd – ferredoxin; FMN – flavin mononucleodide; Fe/S cluster – iron-sulfur cluster; FNR – ferredoxin: NADP+ oxidoreductase; GAT – glutamine amidotransferase; GDH – glutamate dehydrogenase; GOGAT – glu- tamate synthase; GLN1(2) – cytosolic (chloroplastic) glutamine synthetase gene; glsF – ferredoxin-gluta- mate synthase gene; GLT – NADH-glutamate synthase gene; gltB – NADPH-glutamate synthase a subunit 192 gene; gltD – NADPH-glutamate synthase b subunit gene; GltS – glutamate synthase; gltS – Fd-glutamate synthase gene; GLUI (2) – ferredoxin-glutamate synthase 1(2) gene; GS1(2) – cytosolic (chloroplastic/ plastidial) glutamine synthetase; NAR – nitrate reductase gene; NIR – nitrite reductase gene; Rubisco – ribulose-l,5-bisphosphate carboxylase/oxygenase Introduction bacteria (Reitzer 1996). Both the Fd-glutamate synthase and NADH-glutamate synthase are Plants utilize inorganic nitrogen in the form of located in the chloroplast or plastid (Oliveira et al. ) + nitrate (NO3 ) and ammonium ion (NH4 ), when 1997). GS and glutamate synthase occur in mul- the latter is available in the soil or from the sym- tiple forms encoded by distinct genes (Lam et al. biotic fixation of atmospheric dinitrogen (N2) into 1996). Although there is some redundancy of + NH4 in root nodules of leguminous species. function among the multiple enzyme forms, for the Nitrate is reduced to nitrite, in a NAD(P)H- most part each form of GS and glutamate synthase dependent reaction, catalyzed by nitrate reductase plays a distinct physiological role in vivo during (NADH-NAR, EC 1.6.6.1; NAD(P)H-NAR, EC nitrogen absorption in roots, N2-fixation in root ) 1.6.6.2; NADPH-NAR, EC 1.6.6.3) in the cytosol. nodules, primary NO3 reduction, photorespira- + Nitrite is subsequently reduced to NH4 in a fer- tory nitrogen cycling and nitrogen translocation redoxin (Fd)-dependent reaction catalyzed by (Vance et al. 1994; Lam et al. 1996). Fd-dependent nitrite reductase (NIR, EC 1.6.6.4) An alternative pathway for the formation of in the chloroplast or plastid. Ammonium ion is the glutamate involves the reductive amination of + final form of inorganic nitrogen and the nitrogen 2-oxoglutarate by NH4 , catalyzed by mitochon- present in all organic nitrogen compounds, such as drial glutamate dehydrogenase (NADH-GDH, EC amino acids and nucleic acids, is derived from 1.4.1.2). However, the role of GDH in plant cells + NH4 (Lea et al. 1990). Ammonium is released remains controversial (Fox et al. 1995; Melo- and then re-assimilated during nitrogen mobiliza- Olivera et al. 1996; Miflin and Habash 2002). tion in germinating seeds, during the photorespi- Molecular genetic and biochemical studies using ratory conversion of glycine to serine in the cells of 13N- or 15N-radiorabeled tracers, enzyme inhibi- growing leaf, and during nitrogen remobilization tors, and mutants, as well as studies using trans- from sources to sinks (Ireland and Lea 1999). The genic plants affected in GS, glutamate synthase or + assimilation of NH4 into glutamine and gluta- GDH all indicate that the GS/glutamate synthase + mate is the crucial step in amino acid synthesis and cycle is the primary pathway for NH4 assimila- nitrogen metabolism. Glutamine synthetase (GS, tion (Ratcliffe and Shachar-Hill 2001; Lea and + EC 6.3.1.2) catalyzes the first step of NH4 Miflin 2004). Also, expression analysis revealed incorporation into glutamate using ATP to yield that the plants display cell-specific and organ- glutamine in the cytosol (GS1), in chloroplasts and specific patterns for expression of GS and gluta- in plastids (GS2). mate synthase genes by sensing the light and Glutamate synthase (glutamine: 2-oxoglutarate metabolite signals in the regulation of in vivo amidotransferase, henceforth abbreviated as either function of GS and glutamate synthase isoforms GOGAT or GltS) transfers the amide-nitrogen of (Edwards et al. 1990; Thum et al. 2003). The L-glutamine to 2-oxoglutarate, providing two amino-nitrogen of glutamate, incorporated into molecules of L-glutamate. Glutamate synthase in the carbon skeleton by the sequential reaction of plants is present in two distinct forms, one that GS and glutamate synthase, then serves as the uses reduced ferredoxin as the electron donor (Fd- source of the amino groups of aspartate and ala- GOGAT/Fd-GltS, EC 1.4.7.1) and one that uses nine, formed by the transamination of oxaloace- NADH as the electron donor (NADH-GOGAT/ tate and pyruvate, respectively (Reitzer 1996). As NADH-GltS, EC 1.4.1.14). A third form, which amino acid synthesis is controlled by availability uses NADPH as the electron donor (NADPH- of carbon skeletons, nitrogen assimilation is tightly GOGAT/NADPH-GltS, EC 1.4.1.13) is found in coupled to carbon metabolism. Glutamate, 193 aspartate and alanine then provide the nitrogen endosperms (Oaks et al. 1979), roots (Oaks et al. required for the formation of other amino acids. 1979), root nodules (Chen and Cullimore 1988) The amide-nitrogen of glutamine is used for the and cultured soybean cells (Chiu and Shargool biosynthesis of amino acids, including the forma- 1979). Although the results of these biochemical tion of asparagine from aspartate (Ireland and Lea studies suggest the presence of NADPH-glutamate 1999). Glutamine, asparagine, glutamate and synthase in these tissues, NADPH-glutamate syn- aspartate are the major amino acids in leaves and thase protein has not yet been unambiguously roots and are transported in the vascular tissues to identified in either photosynthetic or non-photo- control the nitrogen status during growth and synthetic tissues of any higher plant. It should also development of plants (Pate and Layzell 1990). be pointed out that no open reading frame coding In this review, we will analyze the current for NADPH-glutamate synthase has been detected information on the distribution of different types in the Arabidopsis genome database. of glutamate synthase in prokaryotes and Fd-glutamate synthase and NADH-glutamate eukaryotes. As reaction mechanisms and struc- synthase from the green alga Chlamydomonas tural aspects of glutamate synthase will be dis- reinhardtii have both been characterized (Galva´n cussed in the accompanying article by Vanoni et al. 1984; Ma´rquez et al. 1984). Fd-glutamate et al., we will present instead a summary of the synthase has been detected in the chloroplast of current state of knowledge of the oxidation- green alga Caulerpa simpliciuscula (McKenzie reduction
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